Gene expression in human adipose-derived stem cells: comparison of 2D films, 3D electrospun meshes or co-cultured scaffolds with two-way paracrine effects.

Finding the appropriate cues to trigger the desired differentiation is a challenge in tissue engineering when stem cells are involved. In this regard, three-dimensional environments are often compared to cells' two-dimensional culture behaviour (plastic culture dish). Here, we compared the gene expression pattern of human adipose-derived stem cells (ASC) seeded in a three-dimensional (3D) electrospun mesh and on a two-dimensional (2D) film - both of exactly the same material. Additionally, we conducted experiments with a scaffold floating above a film to investigate two-way paracrine effects (co-system). Electrospun meshes (3D scaffolds) and films (2D), consisting either of pristine poly-lactic-co-glycolic acid (PLGA) or of PLGA-containing dispersed amorphous calcium phosphate nanoparticles (PLGA/aCaP), were seeded with ASCs and cultured either in Dulbecco Minimum Essential Medium (DMEM) or in osteogenic medium. After two weeks, minimum stem cell criteria markers as well as typical markers for osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analysed by quantitative real-time PCR. Interestingly, mostly osteogenic genes of cells seeded on 3D meshes were upregulated compared to those on 2D films, while stem cell markers seemed to be only slightly affected. Runx2 and osteocalcin showed an especially strong upregulation under all conditions, while most other factors analysed for 2D/3D changes were highly dependent on the material composition, the culture medium and on paracrine signalling effects. The beneficial 3D environment for stem cells found in many studies has therefore not to be attributed to the third dimension alone and should carefully be compared to 2D films fabricated of the same material. Furthermore, paracrine interactions triggering differentiation are not negligible.